Hi
I have been trying to make a GFP fusion construct (N-terminal). So basically i fused the 5'UTR region of the gene of interest and fused in to GFP in frame under a pBAD promoter. Both the pBAD promoter and the GFP protein was already available in the starting plasmid. All i did was PCR amplify the region of interest and inserted between the promoter and GFP. The plasmid was sequenced and it seems fine. But when i induced for GFP expression (arabinose) i don't see GFP expression compared to un-induced cells. I really don't what to make of it. Has anyone experienced this type of problem before? What could be the problem?
Thanks.

-nilsh450-

Did you check where the protein localises? I mean if you have a clue from its signal peptide. I maybe wrong, but you may not see anything if it's transported into cytosol and that is the big problem of GFP.

-aces-

it is possible that your fusion partner prevents the GFP from correct folding ...you did SDS-PAGE or western to confirm your fusion protein is made upon induction?